PROJECT SUMMARY Although tobacco use remains the leading preventable cause of disease and death in the United States, all tobacco products do not confer similar public health risk. Specifically, combusted products tend to be viewed as more harmful than non-combusted products. One research priority for the Food and Drug Administration (FDA) is to better understand product toxicity including the development and validation of new biomarkers related to non-cigarette tobacco exposure, harm, or toxicity. Biomarkers that are able to distinguish between types of product use are vital to track the impact of regulatory action and associated proximal and distal health effects related to product use. The goal of this study is to measure nicotelline, a minor tobacco alkaloid associated with tobacco smoker particulate matter, in urine biospecimens from Wave 1 of the Population Assessment of Tobacco and Health (PATH) Study. Aims include to validate nicotelline and its utility (alone or in combination with other biomarkers of tobacco exposure) to distinguish combusted product use (i.e., cigarettes) from non-combusted product use (i.e., smokeless tobacco [SLT] and electronic cigarettes [e-cigarettes]). Previous research has shown when taken as a ratio with its parent compound (anatalline), nicotelline has shown promise in distinguishing SLT use from combusted tobacco use. Therefore, we propose to expand on this existing literature by measuring these compounds in a large sample (N= 140 each) of exclusive daily SLT users, exclusive daily cigarette smokers, and dual SLT + cigarette users to: (1a) validate anatalline/nicotelline ratio to distinguish exclusive SLT users from exclusive cigarette smokers, and (1b) determine anatalline/nicotelline ratio cut-points to distinguish exclusive SLT, dual SLT + cigarette use, and exclusive cigarette use. In addition, we propose exploring if this biomarker may also be able to differentiate other non- combusted product use, specifically e-cigarettes, from combusted product use. We propose additional specific aims among exclusive e-cigarette users (N= 140), and dual e-cigarette + cigarette users (N= 140): (2a) measure nicotelline to distinguish exclusive e-cigarette users from exclusive cigarette smokers, and (2b) determine cut-points of nicotelline and ratios of nicotelline-to-traditional tobacco biomarkers (i.e., 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL]) to distinguish exclusive e-cigarette, dual e-cigarette + cigarette use, and exclusive cigarette use. All aims will be evaluated by comparing levels of exposure using nonparametric Kruskal-Wallace analysis of variance (Aim 1a, 2a) and calculating Receiver Operating Curve (ROC) characteristics (Aim 1b, 2b) to determine cut-points for distinguishing different tobacco product use. Based on biospecimen availability online, https://www.icpsr.umich.edu/icpsrweb/content/NAHDAP/pathstudy- biospec-index.html, these specimens should be available in sufficient quantities. Given the diversity of tobacco products and the different patterns of product use, new tools are needed to help discriminate use of one tobacco product from another and also to identify patterns of polytobacco use.